1. Field of the Invention
The present invention relates to a novel protein showing both phospholipase and lipase activities, more precisely a gene isolated from the microbial metagenome of tidal flat sediment and displaying both phospholipase and lipase activities and a protein encoded therefrom showing the coactivity of calcium-dependent phospholipase and lipase.
2. Description of the Related Art
Lipase (glycerol ester hydrolase, EC 3.1.1.3) is a carboxy ester hydrolase belonging to α/β hydrolases that are able to decompose or synthesize long-chain acylglycerol. Up to date, a variety of animals, plants, and microorganisms have been confirmed to synthesize lipase. Accordingly, studies on the biochemical characteristics of lipase and on lipase genes have been actively undergoing. The endogenous lipase is not only involved in fat metabolism but also comparatively stable in organic solvents. This endogenous lipase does not need coenzymes, has wide substrate specificity and comparatively high optical specificity, making it an excellent biocatalyst for bioconversion, in the field of detergent industry, food additive production, pitch elimination in paper industry, and others. Studies have been under-going to mass-produce such industrially useable and valuable lipase with high efficiency. In particular, studies have been focused mainly on microorganisms producing lipase. Hosts capable of producing lipase are exemplified by Candida sp., Bacillus sp., Penicilium sp., Mucor sp., Rhizopus sp., Pseudomonas sp., and Streptomyces sp.
Lysophospholipid is generated from hydrolysis of phospholipid by phospholipase, which not only acts like a functional group in the course of platelet aggregation but also mediates various physiological activities including signal transduction or plays a role in preventing over-ripen of fruits and plants as a plant hormone. In particular, lysophospholipid has a high water-solubility and can form a stable emulsion even at different hydrogen ion concentrations and temperatures. lysophospholipid is also stable in the presence of magnesium and calcium ions, so that it has been used in the fields of medicine, cosmetics, and food industry.
The said lysophospholipid can be generated from phospholipid mediated by phospholipase in a certain biochemical pathway and at this time phospholipase A hydrolyzes 1-acyl group or 2-acyl group of phospholipid to produce lysophospholipid and fatty acid. This phospholipase A is an essential enzyme in the synthesis of phospholipid containing useful fatty acid such as polyunsaturated fatty acid (PUFA) exemplified by DHA or EPA, etc. This phospholipase A is isolated from various mammals, snake or bee venom, and microorganisms such as Serratia sp., Aspergillus sp., Streptomyces sp., and Fusarium sp. and can be applied to food industry. In order for this enzyme to be used in more industrial fields, substrate specificity or enzyme stability of this enzyme has to be improved (De Maria et al., Appl. Microbiol. Biotechnol. 74:290-300, 2007).
Both lipase and phospholipase display similar mechanism to each other. However, lipase obtained from Staphylococcus hyicus is the only enzyme displaying coactivity to lipid and phospholipid (van Oort et al., Biochemistry, 28:9278-9285, 1989). The enzyme originated from S. hyicus is hard to be produced in a large scale and has comparatively low stability, which makes it less usable in industry.
In the field of fine chemistry producing high value-added lead compounds including medicinal products, when ester compounds are synthesized by the conventional chemical method, the synthesis is achieved at high temperature under high pressure with requiring high consumption of energy, which causes many side reactions that might have a bad effect on the quality of the product. In addition, the conventional method has disadvantages of low conversion rate and low purify in some optical isomers, because of which the production of high purity fine chemical product has been troubled. To overcome the above problem, recent studies have been focused on taking advantage of such reaction that uses the enzyme displaying site specificity and optical specificity as a biocatalyst. However, this attempt has been limited in its application because of the problem of losing the enzyme activity at low temperature.
Lipase hydrolyzes lipid dirt into water-soluble fatty acid or glycerol, suggesting that it makes the function of a surfactant easy. So, lipase has been a target as a detergent or a bleach additive, which was not practical so far, though. This is because lipase loses its enzyme activity at a low washing temperature, meaning oil and fat components are not eliminated completely.
The microorganisms suitable for culture were the major targets of the attempt to find out an enzyme having excellent activity and stability. Various enzymes identified from some of those microorganisms have been used industrially. However, recent molecular-microbial ecology studies proved that at least 99% microorganisms in the natural world are not separated or identified either by the conventional culture method performed in a lab (Amann et al., Microbiol. Rev. 59: 143-169, 1995; Hugenholtz and Pace, Trends Biotechnol. 14: 190-197, 1996; Ward et al., Nature 345: 63-65, 1990). Therefore, a new attempt has been made to search novel genes that could not been identified because of the difficulty in culture from the library constructed by using metagenome, the genome of the microorganisms extracted directly from the natural world without the process of culture and further to develop useful materials therefrom.
Metagenome is the definition indicating the genome of all microorganisms existing in the natural world. In general, the metagenome study is composed of the following steps; isolating metagenome from microorganisms in the natural world without culture; constructing library thereof; and introducing the library into E. coli suitable for culture. This method is to obtain useful materials from those microorganisms which could not be cultured. Even though it is very hard to obtain information about such microorganism, the origin of a target gene, this method has the advantage of obtaining the useful product and gene of the microorganism at the same time.
A research team at University of Wisconsin, USA, was the first study group who succeeded in isolation of massive metagenome and thereafter constructed metagenome library by cloning the metagenome into bacterial artificial chromosome (BAC) vector. They also succeeded in isolation of broad spectrum antibiotics and the genes involved therein (Gillespie et al., Appl. Environ. Microbiol. 68: 4301-4306, 2002; Rondon et al., Appl. Environ. Microbiol. 66: 2541-2547, 2000). A TIGR (The Institute for Genomic Research) team also constructed the general marine microorganism metagenome library in BAC vector to screen genetic resources of those marine microorganisms that could not be cultured so far.
The present inventors isolated a novel gene from the microbial metagenome library obtained from the tidal flat sediment where have a unique microbial diversity including the various unculturable microorganisms, constructed a vector containing the said gene, transfected E. coli with the vector, and accordingly confirmed that the protein produced from the transformant constructed above displayed excellent phospholipase and lipase activities together and had excellent activity and stability as well even at a low temperature and in alkali condition, leading to the completion of the present invention.